博碩士論文 952211004 詳細資訊




以作者查詢圖書館館藏 以作者查詢臺灣博碩士 以作者查詢全國書目 勘誤回報 、線上人數:10 、訪客IP:18.232.99.123
姓名 王思寰(Szu-Huan Wang)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 人類陰道滴蟲之Myb2蛋白質動態性質研究
(Probing the Protein Dynamics of Myb2 from Human Trichomonas vaginalis)
相關論文
★ 分析原核生物基因體複製起點與終點的反向對偶對稱現象★ 分析基因體拷貝數變異所使用的兩種方法比較:隱藏馬可夫模型與成對高斯合併法
★ 使用兩種方法偵測基因體拷貝數變異:成對高斯合併法與隱藏馬可夫模型★ 以整體晶片數據為母體應用於分析基因差異表達的z檢定方法
★ GSLHC - 運用基因組及層次類聚以生物功能群將有生物活性的複合物定性的方法★ 一個檢定測量微晶片基因表達數據靈敏度的全統計計算法
★ 運用嶄新抗體固著策略發展及驗證新式抗體微晶片平台★ Drug-resistant colon cancer cells produce high carcinoembryonic antigen and might not be cancer-initiating cells
★ 創傷性關節炎軟骨之退化進程- 大鼠模型基因體圖譜研究★ 運用時間序列微陣列資料來預測調控基因
★ 以大鼠嗜鉻性瘤細胞株建立神經訊號傳遞之細胞分子生物學模型★ 一種找尋再利用藥物複合物來系統性治療複雜疾病的架構:大腸直腸腺瘤的應用
★ 以上皮細胞間質化與增生相關功能來描述癌症幹細胞之基因型★ 從共表達差異基因對導出正常腦老化及因阿茲海默症特定腦區導致在功能性基因途徑與樞紐基因子網絡之變化
★ 以疾病進展趨勢挑選基因法識別正常腦老化與阿爾茨海默氏症在特定腦區引發的關鍵功能路徑與調節路徑之變化★ 中國傳統醫藥蒙古黃耆在HCT116結腸癌細胞體外和體內實驗呈現腫瘤抑制作用
檔案 [Endnote RIS 格式]    [Bibtex 格式]    [相關文章]   [文章引用]   [完整記錄]   [館藏目錄]   至系統瀏覽論文 ( 永不開放)
摘要(中) 陰道毛滴蟲(Trichomonas vaginalis)是一種厭氧性的寄生單細胞原蟲,具有鞭毛,主要寄生於女性的陰道、尿道、子宮,以及男性的前列腺,進而引起滴蟲性陰道炎,此疾病為世界最普遍的直接性接觸感染疾病。目前研究發現,致病的關鍵其中為黏附蛋白質所產生的細胞黏附作用。而基因ap65-1是黏附蛋白質(ap65)家族的一員,分子量為65 道爾吞。調控基因ap65-1的轉錄因子被認為是陰道毛滴蟲內的Myb1與Myb2蛋白質。
在基因ap65-1上有兩段DNA序列可供Myb蛋白質所辨認,分別為MRE1/MRE2r與MRE2f ,進而調控此基因的轉錄機制。研究中發現陰道毛滴蟲內的Myb2蛋白質可以與DNA序列MRE1/MRE2r與MRE2f結合;利用分子生物學的方法截取全長Myb2蛋白質中胺基酸序列從V40到M156的Myb2x截斷蛋白質,且保有與全長Myb2蛋白質相似的DNA結合能力。
在此,利用核磁共振的技術去探討截斷蛋白Myb2x以及分別與DNA序列MRE1/MRE2r與MRE2f結合後在分子動態上的差異性。在14.7Tesla的靜磁場下,藉由使用異核核磁共振脈衝序列量測遲緩速率(relaxation rate)和15N-1H異核交互作用增異參數(NOE);並利用簡化光譜密度(reduced spectral density mapping)得到光譜密度函數再與結構比較蛋白質的分子動態差異性。未來,將可以繼續探討Myb2x蛋白質更多的詳細結合機制,進而在設計藥物上有所幫助。
摘要(英) Trichomonas vaginalis, an anaerobic, parasitic flagellated protozoan resides mostly in female vagina, urethra, uterus as well as male prostate gland, is the causative agent of the most common nonviral sexually transmitted infections (STIs) in the world, human trichomoniasis. Recently, multiple surface adhesion proteins have been shown to be engaged in Cytoadherence, an essential step, of the T. vaginalis infection. The ap65-1 gene, a member of the adhesion protein 65 (ap65) multigene family, encodes for multiple homologous 65-kDa proteins, was found to be regulated by transcription factors, Myb1 and Myb2 proteins, in T. vaginalis.
Two DNA sequences, MRE1/MRE2r and MRE2f, on the gene, ap65-1, is recognized by Myb protein; this discovery deduce the possible involvement of Myb-like transcription factors related to the transcription mechanism within T. vaginalis. From various experiments, evidences show a specific interaction between full-length Myb2 protein with MRE1/MRE2r and MRE2f. We have found that a truncated fragment of Myb2, designated as Myb2x, consisting of amino acid V40–M156 displays similar DNA affinity. This fragment was employed for affinity binding and NMR structure-dynamic studies.
By NMR relaxation technology, the difference in dynamics between Myb2x free, Myb2x-MRE1/MRE2r and Myb2x- MRE2f complex forms was resolved. 15N spin relaxation rates and heternuclear (15N-1H) NOE were measured by standard pulse sequences at static magnetic field of 14.7 Tesla. The relaxation data were further analyzed with reduced spectral density mapping approach to deduce the spectral density functions: J(0), J(ωN) and J(ω0.87H) . The protein dynamics of Myb2x, as revealed by the reduced spectral density functions, are mapped onto the structures of the free and DNA-bound forms of Myb2x. The results showed that binding of DNA tighten the protein structure considerably. Such information will be useful for design of effective drugs for the treatment of human trichomoniasis.
關鍵字(中) ★ 核磁共振
★ 陰道滴蟲
★ Myb2蛋白質
★ 動態性質
關鍵字(英) ★ NMR
★ Trichomonas vaginalis
★ Myb2 protein
★ Dynamics
論文目次 摘要 Ⅰ
Abstract Ⅱ
誌謝 Ⅳ
Content Ⅴ
Figure and Table list Ⅷ
1. Introduction
1.1 Trichomonas Vaginalis 1
1.2 Adhesion Protein 2
1.3 Myb Protein 3
1.4 Specific Aim 4
2. Theory
2.1 Introduction to NMR 7
2.2 Introduction to Relaxation 9
2.2.1 Origin of relaxation 10
2.2.2 Random fields 11
2.2.2.1 Chemical shift anisotropy 12
2.2.2.2 Dipole-dipole interaction 14
2.2.3 Relaxation parameters: R1 and R2 and NOE 15
2.2.3.1 Relaxation parameters: R1 and R2 15
2.2.3.2 Relaxation parameters: NOE and Solomon's equation 16
2.3 Correlation Function and Spectral Density Function 19
2.4 Reduced Spectral Density Mapping 21
2.5 1D NMR Spectra of A Protein 23
2.6 Circular Dichroism 24
3. Experimental Design and Methods
3.1 Protein Preparation and Purification 30
3.1.1 Preparation of Myb2x protein 30
3.1.2 Preparation of Myb2x protein with N15-label 31
3.1.3 Preparation of N15-labeled Myb2x protein for Fe2+ binding 31
3.1.4 Preparation of DNA for DNA binding 32
3.2 Protein Information Detecting 32
3.2.1 SDS-PAGE 32
3.2.2 Ion exchange chromatography 32
3.2.3 Gel filtration chromatography 33
3.3 Circular Dichrosim (CD) Experiment 33
3.4 Data Acquisition and Process 34
3.5 Reduced Spectral Mapping 36
4. Results
4.1 Prediction of Myb2 Protein 38
4.2 Purification and SEC Profile of Myb2x Protein 39
4.3 Characterization of Myb2x Protein 40
4.3.1 Circular dichroism (CD) analysis of Myb2x protein 40
4.3.2 NMR analysis of free and bound forms of Myb2x protein 40
4.3.3 Interaction for Myb2-MRE1/MRE2r and Myb2-MRE2f
bound forms and Fe2+ iron by NMR analysis 42
4.4 Measure Relaxation Parameters 43
4.5 Estimation of Overall Correlation Time 45
4.6 Dynamic Parameters 45
5. Discussion 64
6. Future work 67
Acknowledgement 68
References 68
參考文獻 1. Soper, D. "Trichomoniasis: under control or undercontrolled?" American Journal of Obstetrics and Gynecology Vol. 190, pp. 281-290 (2004).
2. Peter, U. & Jacqueline, A.U. "Drug targets and mechanisms of resistance in the anaerobic protozoa". Clinical Microbiology Reviews Vol. 14, pp. 150-164 (2001).
3. V. Moreno-Brito et al. "A Trichomonas vaginalis 120 kDa protein with identity to hydrogenosome pyruvate:ferredoxin oxidoreductase is a surface adhesin induced by irony iron". Cellular Microbiology Vol. 7, pp. 245-258 (2005).
4. J.M. Carlton et al. "Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis". Science Vol. 315, pp. 207-212 (2007).
5. J. R. Schwebke & D. Burgess. "Trichomoniasis". Clinical Microbiology Reviews Vol. 17, pp. 794-803 (2004).
6. BonDurant, R.H. "Diagnosis, treatment and control of bovine trichomoniasis". Compendium for Continuing Education for the Practicing Veterinarian Vol. 7, pp. 179-188 (1985).
7. J. P. Heath. "Behaviour and pathogenicity of Trichomonas vaginalis in epithelial cell cultures: A study by light and scanning electron microscopy". British Journal of Venereal Diseases Vol.57, pp. 106-117 (1981).
8. Patricia C. Guenthner, W. Evan Secor & Charlene S. Dezzutti. "Trichomonas vaginalis-induced epithelial monolayer disruption and human immunodeficiency virus type 1 (HIV-1) replication: Implications for the sexual transmission of HIV-1". Infection and Immunity Vol. 73, pp. 4155-4160 (2005).
9. Rughooputh, S. & Greenwell, P. "Trichomonas vaginalis : paradigm of a successful sexually transmitted organism". British Journal of Biomedical Science Vol. 62, pp. 193-200 (2005).
10. D. Sobel, P. Nyirjesy & William Brown. "Tinidazole therapy for metronidazole-resistant vaginal Trichomoniasis". Clinical Infectious Diseases Vol. 33, pp. 1341-1346 (2001).
11. Karen A. Wendel & Kimberly A. Workowski. "Trichomoniasis: Challenges to appropriate management". Clinical Infectious Diseases vol. 44, pp. 123-129 (2007).
12. Jane R. Schwebke & Frank J. Barrientes. "Prevalence of Trichomonas vaginalis Isolates with Resistance to Metronidazole and Tinidazole". Antimicrobial Agents and Chemotherapy Vol. 50, pp. 4209-4210 (2006).
13. V. Mundodi, A.S. Kucknoor, T-H Chang & J.F. Alderete. "A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells". BMC Microbiology Vol. 6(2006).
14. R. Arroyo, J. Engbring & J. F. Alderete. "Molecular basis of host epithelial cell recognition by Trichomonas vaginalis". Molecular Microbiology Vol. 6, pp. 853-862 (1992).
15. V. Mundodi, A. S. Kucknoor, D. J. Klumpp, T. -H. Chang & J. F. Alderete. "Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis". Molecular Microbiology Vol. 53, pp. 1099-1108 (2004).
16. Ana F. Garcia, Te-Hung Chang, Marlene Benchimol, Michael W. Lehker & John F. Alderete. "Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis". Molecular Microbiology Vol. 47, pp. 1207-1224 (2003).
17. Casta e Silva Filho F, de Souza W & Lopes JD. "Presence of laminin-binding proteins in trichomonads and their role in adhesion". Cell Biology Vol. 85, pp. 8042-8046 (1988).
18. Kathy Weston. "Myb proteins in life, death and differentiation". Current Opinion in Genetics & Development Vol. 8, pp. 76-81 (1998).
19. Lipsick, J.S. "One billion years of Myb". Oncogene Vol. 13, pp. 223-235 (1996).
20. K. M. Howe, C. F. Reakes & R.J.Watson. "Characterization of the sequence-specific interaction of mouse c-myb protein with DNA". The EMBO Journal Vol. 9, pp. 161-169 (1990).
21. B. Lüscher & R. N. Eisenman. "New light on Myc and Myb. Part II. Myb." Genes & Development Vol. 4, pp. 2235-2241 (1990).
22. H. Sakura et al. "Delineation of three functional domains of the transcriptional activator encoded by the c-myb protooncogene". Proc Natl Acad Sci U S A. Vol. 86, pp. 5758-5762 (1989).
23. Biedenkapp, H., Borgmeyer, U., Sippel, A.E. & Klempnauer, K.H. "Viral myb oncogene encodes a sequence-specific DNA-binding activity". Nature Vol. 335, pp. 835-837 (1988).
24. J. Tanikawa et al. "Recognition of specific DNA sequences by the c-myb protooncogene product: role of three repeat units in the DNA-binding domain". Proc Natl Acad Sci U S A. Vol. 90, pp. 9320-9324 (1993).
25. Kathy M. Howe & Roger J. Watson. "Nucleotide preferences in sequence-specific recognition of DNA by c-myb protein". Nucleic Acids Research Vol. 19, pp. 3913-3919 (1991).
26. S.J. Ong, H.M. Hsu, H.W. Liu, C.H. Chu & J.H. Tai. "Multifarious transcriptional regulation of adhesion protein gene ap65-1 by a novel Myb1 protein in the protozoan parasiteTrichomonas vaginalis". Eukaryotic Cell Vol. 5, pp. 391-399 (2006).
27. S.J. Ong, H.M. Hsu, H.W. Liu, C.H. Chu & J.H. Tai. "Activation of multifarious transcription of an adhesion protein ap65-1 gene by a novel Myb2 protein in the protozoan parasite Trichomonas vaginalis". Journal of Biological Chemistry Vol. 282, pp. 6716-6725 (2007).
28. Akke, M. "NMR methods for characterizing microsecond to millisecond dynamics in recognition and catalysis". Current Opinion in Structural Biology Vol. 12, pp. 642-647 (2002).
29. Palmer, A.G., 3rd, Kroenke, C.D. & Loria, J.P. "Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules". Methods Enzymol Vol. 339, pp. 204-238 (2001).
30. Palmer, A.G., 3rd. "NMR characterization of the dynamics of biomacromolecules". Chemical Reviews Vol. 104, pp. 3623-3640 (2004).
31. Levitt, M.H. Spin Dynamics - Basics of Nuclear Magnetic Resonance. John Wiley & Sons, Ltd (2001).
32. Keeler, J. Understanding NMR Spectroscopy. John Wiley & Sons, Ltd (2005).
33. Palmer, A.G., Rance, M. & Wright, P.E. "Intramolecular motions of a zinc finger DNA-binding domain from Xfin characterized by proton-detected natural abundance 13C heteronuclear NMR spectroscopy". Journal of the American Chemical Society Vol. 113, pp. 4371-4380 (1991).
34. Palmer, A.G. & Case, D.A. "Molecular dynamics analysis of NMR relaxation in a zinc-finger peptide". Journal of the American Chemical Society Vol. 114, pp. 9059-9067 (1992).
35. Cavanagh, J., Wayne, J.F., Palmer, A.G., 3rd, Skelton, N.J. & Rance, M. Protein NMR Spectroscopy Principles and Practice. Academic Press (2006).
36. Evans, J.N.S. Biomolecular NMR Spectroscopy. Oxford University Press (1995).
37. Norma H. Pawley, Chunyu, W., Koide, S. & Nicholson, L.K. "An improved method for distinguishing between anisotropic tumbling and chemical exchange in analysis of 15N relaxation parameters". Journal of Biomolecular NMR Vol. 20, pp. 149-165 (2001).
38. Queen's University, Advanced NMR course for the Web. In;. http://www.chem.queensu.ca/facilities/NMR/nmr/webcourse/index.htm.
39. N. Bloembergen, Purcell, E.M. & Pound, R.V. "Relaxation effects in nuclear magnetic resonance absorption". Physical Review Vol. 73, pp. 679-712 (1948).
40. Peng, J.W. & Wagner, G. "Mapping of spectral density function using heteronuclear NMR relaxation measurements". Journal of Magnetic Resonance Vol. 98, pp. 308-332 (1992).
41. Abragam, A. Principles of Nuclear Magnetism Oxford University Press (1983).
42. Peng, J.W. & Wagner, G. "Mapping of the spectral densities of N-H bond motions in eglin c using heteronuclear relaxation experiments". Biochemistry Vol. 31, pp. 8571-8586 (1992).
43. Dayie, K.T., Wagner, G. & Lefevre, J.F. "Theory and practice of nuclear spin relaxation in proteins". Annual Review of Physical Chemistry Vol. 47, pp. 243-282 (1996).
44. Farrow, N.A., Zhang, O., Szabo, A., Torchia, D.A. & Kay, L.E. "Spectral density function mapping using 15N relaxation data exclusively". Journal of Biomolecular NMR Vol. 6, pp. 153-162 (1995).
45. Atkinson, R.A. & Kieffer, B. "The role of protein motions in molecular recognition: insights from heteronuclear NMR relaxation measurements". Progress in Nuclear Magnetic Resonance Spectroscopy Vol. 44, pp. 141-187 (2004).
46.http://www.newark.rutgers.edu/chemistry/grad/chem585/lecture1.html.
47. Barbato, G., Ikura, M., Kay, L., Pastor, R. & Bax, A. "Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible". Biochemistry Vol. 31, pp. 5269-5278 (1992).
48. Kay, L.E., Nicholson, L., Delaglio, F., Bax, A. & Torchia, D. "Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins". Journal of Magnetic Resonance Vol. 97, pp. 359-375 (1992).
49. Wishart, D.S. & Sykes, B.D. "Chemical shifts as a tool for structure determination". Methods of Enzymology Vol. 239, pp. 363-392 (1994).
50. Wishart, D.S. et al. "1H, 13C and 15N chemical shift referencing in biomolecular NMR". Journal of Biomolecular NMR Vol. 6, pp. 135-140 (1995).
51. Piotto, M., Saudek, V. & Sklenar, V. "Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions". Journal of Biomolecular NMR Vol. 2, pp. 661-665 (1992).
52. Sklenar, V., Piotto, M., Leppik, R. & Saudek, V. "Gradient-tailored water suppression for 1H-15N HSQC experiments optimized to retain full sensitivity". Journal of Magnetic Resonance Vol. 102A, pp. 241-245 (1993).
53. Farrow, N.A. et al. "Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation". Biochemistry Vol. 33, pp. 5984-6003 (1994).
54. Markley, J.L., Horsley, W.J. & Klein, M.P. "Spin-lattice relaxation measurements in slowly relaxing complex spectra". Journal of Chemical Physics Vol. 55, pp. 3604-3605 (1971).
55. Neidig, K.P. et al. "AURELIA, a program for computer-aided analysis of multidimensional NMR spectra". Journal of Biomolecular NMR Vol. 6, pp. 255-270 (1995).
56. Kroenke, C.D., Rance, M. & Palmer, A.G. "Variability of the 15N chemical shift anisotropy in escherichia coli ribonuclease H in solution". Journal of the American Chemical Society Vol. 121, pp. 10119-10125 (1999).
57. Koradi, R., Billeter, M. & Wuthrich, K. "MOLMOL: a program for display and analysis of macromolecular structures". Journal of Molecular Graphics Vol. 14, pp. 51-55 (1996).
58. Sasaki, M., Ogata, K., Hatanaka, H. & Nishimura, Y. "Backbone dynamics of the c-Myb DNA-binding domain complexed with a specific DNA". The Journal of Biochemistry Vol. 127, pp. 945-953 (2000).
59. K. Ogata et al. "Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices". Cell Vol. 79, pp. 639-648 (1994).
指導教授 李弘謙、黃太煌
(Hoong-Chien Lee、Tai-Huang Huang)
審核日期 2008-7-10
推文 facebook   plurk   twitter   funp   google   live   udn   HD   myshare   reddit   netvibes   friend   youpush   delicious   baidu   
網路書籤 Google bookmarks   del.icio.us   hemidemi   myshare   

若有論文相關問題,請聯絡國立中央大學圖書館推廣服務組 TEL:(03)422-7151轉57407,或E-mail聯絡  - 隱私權政策聲明